Use Of A Growth-Stimulating Protein

ABSTRACT

This invention relates to the inhibition of a newly discovered growth-stimulating protein in an individual. Further, the invention relates to a method for preventing or treating a cancer, or preventing or treating cancer growth, invasion or metastasis, or preventing or treating other hyperproliferative diseases in an individual, by down regulating the expression of said growth-stimulating protein or by inactivating said protein. Still further, the invention concerns a method for diagnosing cancer or other hyperproliferative diseases in an individual based on said growth-stimulating protein.

FIELD OF THE INVENTION

This invention relates to the inhibition of a newly discoveredgrowth-stimulating protein in an individual. Further, the inventionrelates to a method for preventing or treating a cancer, or preventingor treating cancer growth, invasion or metastasis, or preventing ortreating other hyperproliferative diseases in an individual, by downregulating the expression of said growth-stimulating protein or byinactivating said protein. Still further, the invention concerns amethod for diagnosing cancer or other hyperproliferative diseases in anindividual based on said growth-stimulating protein.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

Cancer is a devastating disease afflicting all communities worldwide. Ithas been estimated that 1 out of 2 men and 1 out 3 women will developsome form cancer within their lifetime. It is also estimated that morethan 550,000 people will die due to cancer in the United States aloneduring 2005. Cancer is a generalized term for a complex and vastlydifferent set of diseases related to the uncontrolled growth, survival,and invasion of cells. Although there are more than 100 different typesof cancer, as well as subtypes of each, recent studies have revealedlimited number of genetic elements which are required for transformationof benign cells to cancer cells (Zhao et al., 2004). Generally, it hasbeen concluded that human cell transformation requires activation of RasGTPase, overexpression of telomerase, inactivation of tumor suppressorproteins p53 and Retinoblastoma protein (Rb) and inhibition of proteinphosphatase 2A (PP2A) (Zhao et al., 2004). It is obvious thatunderstanding of the function of these genetic elements could lead todevelopment of cancer therapies that would be widely applicable todifferent types of cancer.

As described above, inhibition of PP2A activity has been identified asone of the prerequisites for transformation of primary human cells (Zhaoet al., 2004). PP2A is a trimeric protein complex consisting of acatalytic subunit (PP2Ac or C), a scaffold subunit (PR65 or A) and oneof the alternative regulatory B subunits (FIG. 1A) (Janssens and Goris,2001). PP2A regulates cellular behaviour by dephosphorylating regulatoryser/thr residues of the protein kinases and other signaling proteins.Oncogenic transcription factor c-Myc is one of the many substrates forPP2A. It was recently shown, that PP2A-mediated dephosphorylation ofserine 62 on c-Myc results in proteosomal degradation of the protein(Yeh et al., 2004). Importantly, viral small t-antigen, whichinactivates PP2A, exerts its oncogenic potential by stabilization ofc-Myc (Yeh et al., 2004).

Even though the importance of PP2A inhibition for human celltransformation has been firmly established by using viral antigens asresearch tools, the molecular mechanisms by which PP2A inhibition occursin spontaneously transformed human cancer cells is currently notunderstood. There is thus an identified need of elucidating themechanism by which PP2A inhibits transformation.

BRIEF DESCRIPTION OF THE INVENTION

This invention is based on the discovery of a growth-stimulatingprotein. The inventors of the present invention have found that theprotein KIAA1524 (GenBank accession number AA130565; SEQ ID NO. 1) is anendogenous inhibitor of protein phosphatase 2A (PP2A) and that theprotein KIAA1524 is required for tumor growth and cancer cellproliferation and that the protein KIAA1524 is up regulated in cancertissue.

Thus, according to one aspect, this invention concerns a method forscreening and identifying a therapeutic agent, which inhibits KIAA1524,said method comprising the steps of a) providing a first proteinimmobilized in a reaction chamber, b) adding a candidate agent and alabeled second protein to said chamber concomitantly or subsequently inany order, c) determining whether said first protein binds to saidsecond protein, and d) identifying said candidate agent as a therapeuticagent inhibiting KIAA1524 when the determination in step c) is negative,wherein said first protein is KIAA1524 and said second protein isselected from the group consisting of PP2A, subunits thereof, and c-Myc,or vice versa.

According to another aspect, the invention concerns agents inhibitingKIAA1524, such as small interfering RNAs and peptides as described inthe claims. Furthermore, the invention concerns pharmaceuticalcompositions comprising such agents.

According to a further aspect, the invention concerns a method forproducing a pharmaceutical composition, said method comprisingidentifying an agent which inhibits KIAA1524 and mixing said agent withany suitable pharmaceutically acceptable excipient, as well aspharmaceutical compositions produced by such a method.

According to a further aspect, the invention concerns a method ofinhibiting KIAA1524 in a human or animal patient in need thereof byadministering a therapeutically effective amount of a pharmaceuticalcomposition described in the claims.

According to a further aspect, the invention concerns use of an agent,which inhibits KIAA1524 for the manufacture of a pharmaceuticalcomposition for treating, preventing and/or alleviating a diseaseselected from the group consisting of cancer and otherhyperproliferative diseases.

According to a still further aspect, the invention concerns a method fordetermining the invasiveness of a malignant change in a mammal suspectedto suffer from cancer, said method comprising: a) assessing the level ofKIAA1524 expression in a sample, suspected to comprise malignant cells,taken from said mammal, b) comparing the expression level from step a)with the expression level of KIAA1524 in a non-malignant control sample,and c) determining said malignant changes as invasive when theexpression level of KIAA1524 in said sample is significantly higher thanthe expression level of KIAA1524 in a non-malignant control sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. KIAA1524 is an endogenous inhibitor of PP2A. FIG. 1A) shows aschematic picture of the PP2A complex containing PR65 scaffold, PP2Accatalytic subunit and a regulatory B-subunit. TAP-fusion of the PR65protein used in the TAP-purification is shown. FIG. 1B) TAP purificationof the PR65 protein complex from HT-1080 fibrosarcoma cells. Proteins inthe PR65TAP eluates were identified by mass-spectrometric peptidesequencing. FIG. 1C) Co-immunoprecipitation analysis of HeLa cellcytoplasmic extracts with PR65 antibody reveals interaction betweenendogenous KIAA1524, PR65 and PP2Ac proteins. PI, pre-immune serum;input, input material. FIG. 1D) Identification of a mutant of KIAA1524protein deficient in PR65 binding by co-immunoprecipitation analysis.FIG. 1E) HeLa-cells transfected with indicated expression constructswere analyzed by confocal microscopy for KIAA1524-PP2Ac co-localization.FIG. 1F) siRNA-mediated depletion of KIAA1524 protein expression in HeLacells enhances serine/threonine phosphatase activity ofimmunoprecipitated PP2Ac.

FIG. 2. KIAA1524 protein. FIG. 2A) Predicted structure of KIAA1524protein reveals several putative protein-protein interaction domains.KIAA1524mut protein analyzed in FIGS. 1D and 1E. FIG. 2B) Amino acidsequence of the KIAA1524 protein (SEQ ID NO. 1).

FIG. 3. Identification of PP2A interacting domain in KIAA1524. FIG. 3A)Co-immunoprecipation of the KIAA1524 deletion constructs with endogenousPR65 from HeLa cell lysates. Co-immunoprecipitation of indicatedFlag-KIAA1524 proteins and the PP2A complex was analyzed by westernblotting of the anti-Flag immunoprecipitates with the PR65 antibody.Immunoprecipitation efficiency of the KIAA1524 deletions was confirmedby KIAA1524 immunoblotting. FIG. 3B) Identification of putativePP2A-interacting domain in KIAA1524. Results in FIG. 3A show thatwild-type KIAA1524 interacts with PP2A (PR65), whereas KIAA1524 in which461-533 domain is delted displays impaired interaction with PP2A.Therefore, the KIAA1524 domain 461-533 is expected to mediateinteraction between KIAA1524 and PP2A.

FIG. 4. KIAA1524 inhibits c-Myc associated PP2A activity and c-Mycdegradation. FIG. 4A) siRNA-induced depletion of KIAA1524 proteinresults in downregulation of c-Myc protein expression in HeLa cells.FIG. 4B) siRNA-induced depletion of KIAA1524 protein does not inhibitc-Myc mRNA expression. FIG. 4C) siRNA-induced depletion of KIAA1524protein enhances PP2A activity in c-Myc immunoprecipitates. FIG. 4D)KIAA1524 protein does not regulate interaction between PP2Ac and c-Mycproteins. HeLa cells transfected with either scrambled or KIAA1524 siRNAwere subjected to co-immunoprecipitation analysis with c-Myc specificantibody. Proteins in the immunoprecipitates were thereafter studied byindicated antibodies.

FIG. 5. Identification of KIAA1524 interacting domain in c-Myc. FIG. 5A)In vitro binding assay with immobilized, recombinant Flag-KIAA1524protein and recombinant GST protein or with GST-c-Myc 1-262 protein.Immunoblots of eluates with GST (top) or KIAA1524 (bottom). Input, GSTproteins used for interaction assay. Shown is a representative blot ofthree independent experiments with similar results. FIG. 5B) Schematicpresentation of identification of c-Myc 105-262 as KIAA1524 bindingdomain on c-Myc. KIAA1524.

FIG. 6. KIAA1524 is required for cancer cell growth. FIG. 6(A) Thymidineincorporation of HeLa cells transfected for 72 h with scrambled orKIAA1524 siRNA for cell proliferation. Shown is mean+S.D. of fourexperiments. * p<0.05, Student's t-test. FIG. 6B) Flow cytometricanalysis of DNA content for cell cycle progression from HeLa cellstransfected with scrambled or KIAA1524 siRNA for 72 h. Results are froma representative experiment of four repetitions with similar results.FIG. 6C) KIAA1524 depletion does not induce Poly (ADP-ribose) Polymerase(PARP) cleavage in HeLa cells as detected by immunoblotting 72 h afterKIAA1524 siRNA transfection. Expected molecular weights of thefull-length (110 kD) and caspase-cleaved forms of PARP proteins areshown on the right. FIG. 6D) Dense foci formation on a monolayer of HeLacells transfected with scrambled or KIAA1524 siRNA. Above,representative light microscopy images. Below, quantitation of number offoci 10 days after re-plating by Image J software. Shown is average+S.D.of four experiments. * p=0.002, Student's t-test. FIG. 6E) siRNA-induceddepletion of KIAA1524 protein inhibits HeLa cell anchorage independentgrowth on agar. FIG. 6F) HeLa cells transfected with scrambled (Scr) orKIAA1524 siRNA were analyzed for tumor growth in immunocompromisedmouse. Shown is mean+SD of tumor volumes from six mice in each group.FIG. 6G) Weights (mg) of tumors from an independent experiment performedsimilarly to the experiment shown in FIG. 6F) at day 27. * p=0.034,Mann-Whitney U test.

FIG. 7. KIAA1524 is overexpressed in human squamous cell carcinomas ofthe head and neck (HNSCC). FIG. 7A) KIAA1524 mRNA mRNA expression wasquantitated by quantitative RT-PCR analysis from normal tissue samples.KIAA1524 expression is presented relative to β-actin. FIG. 7B) KIAA1524mRNA expression was studied from HNSCC cell lines and from humanepidermal keratinocytes (HEK) by Taqman real-time PCR analysis. FIG. 7C)KIAA1524 mRNA expression was studied from HNSCC tumor samples and fromnormal tissue control samples by Taqman real-time PCR analysis. FIG. 7D)Immunostaining analysis of HNSCC tissue with KIAA1524 antibody showsintense cytoplasmic KIAA1524 staining in tumor cells (tumor cell nodulesindicated by arrows). FIG. 7E) siRNA-induced depletion of KIAA1524protein results in downregulation of c-Myc protein expression in HNSCCcell lines.

FIG. 8. KIAA1524 is overexpressed in human colon cancers. FIG. 8A)Quantitative RT-PCR analysis of KIAA1524 mRNA expression in colon cancertissues and in non-malignant colon tissues (control). Shown is meanexpression of samples+S.D. * p<0.05, Mann-Whitney U test. FIG. 8B)Association between KIAA1524 expression level and tumor gradus in humancolon cancers. Statistical difference in KIAA1524 expression betweengradus III-IV and gradus II (* p<0.0001, Mann-Whitney U test) andbetween gradus III-IV and normal samples (* p=0.0019, Mann-Whitney Utest) was observed.

FIG. 9. KIAA1524 is overexpressed in human breast cancers. FIG. 9A)KIAA1524 expression in normal or tumoral mammary tissue. FIG. 9B)KIAA1524 expression depends on human mammary tumor type. Two-sampleWilcoxon (Mann-Whitney) rank-sum test was used for statistical analysis.*:p<0.05 compared to normal breast; ** p<0.005 compared to mucinoustumors. House-keeping gene β-actin was used for normalization.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on identification of KIAA1524 as agrowth-stimulating protein, which interacts with PP2A and inhibits thetumor suppressor activity thereof. The present invention thus providesKIAA1524 as a target for novel anticancer or antiproliferative agents.

KIAA1524 was identified as a PP2A interacting protein based onco-immunoprecipitation with PR65 protein, a scaffolding subunit of PP2Aprotein complex, and subsequent mass-spectrometric peptide sequencing.The ability of KIAA1524 to interact with PP2A complex was demonstratedby co-immunoprecipiation analysis of endogenous PR65 proteins and byusing deletion mutants of KIAA1524. In addition the functional role ofKIAA1524 as PP2A inhibitor was demonstrated by depleting KIAA1524 byshort interfering RNA (siRNA) oligos targeted to KIAA1524, whichresulted in stimulation of PP2A phosphatase activity.

Furthermore, based on co-immunoprecipitation experiments, KIAA1524 wasidentified as a protein directly interacting with transcription factorc-Myc. Said interaction was shown to stabilize c-Myc protein therebypromoting its oncogenic potential.

The role of KIAA1524 in regulation of cancer cell behavior was studiede.g. by transfecting cells with KIAA1524 siRNA and subsequentlydetermining cell proliferation both in a cell culture and in an in vivomouse model. Depletion of KIAA1524 resulted in inhibition of cellproliferation and inhibition of anchorage independent growth of thecells, as well as compromised tumor formation in mice. Furthermore,KIAA1524 was found to be overexpressed in human malignancies, such ashead and neck squamous cell carcinoma (HNSCC), colon cancer and breastcancer. Especially KIAA1524 overexpression was found in invasive tumorsof colon cancer and breast cancer when compared to non-invasive tumorsof the same origin. These and other results disclosed below indicatethat KIAA1524 promotes cancer cell growth and therefore KIAA1524 servesas a target for cancer therapeutics.

The present invention is directed to a method for screening andidentifying a therapeutic agent which inhibits KIAA1524 and is thususeful in treating, preventing and/or alleviating cancer, cancer cellproliferation, invasion, metastasis, as well as other hyperproliferativediseases, such as psoriasis, myocardial hypertrophy and benign tumor,such as adenoma, hamartoma and chondroma.

As used throughout this specification, the term “inhibiting KIAA1524”includes down-regulating the expression of KIAA1524, inhibiting theactivity of KIAA1524, inactivating KIAA1524, as well as inhibitingKIAA1524 interaction with PP2A complex or c-Myc. The words inhibit andblock, are used interchangeably.

The above method comprises the following steps: a) providing a firstprotein immobilized in a reaction chamber, b) adding a candidate agentand a labeled second protein to said chamber concomitantly orsubsequently in any order, c) determining whether said first proteinbinds to said second protein, and d) identifying said candidate agent asa therapeutic agent inhibiting KIAA1524 when the determination in stepc) is negative but positive in the absence of a therapeutic agent. Inthe method, said first protein is KIAA1524 and said second protein isselected from PP2A, subunits thereof, i.e. PP2Ac α or β, PR65 α or β, orany of the alternative B-subunits (B, B′, B″), and c-Myc, or vice versa.

Immobilization of said first protein to a reaction chamber, such as amulti-well plate, can be performed by any suitable method known in theart. Such methods are apparent to a person skilled who also appreciateshow to immobilize said protein without affecting its conformation orbinding properties.

Any suitable labels and labeling methods known in the art can be usedfor labeling said second protein. Preferably, said label is afluorescent label, such as green fluorescent protein or chemicalfluorescent label such as Texas red. The said label can also be aprotein such as firefly luciferase that emit light when incubated with asubstrate such as luciferin. In addition the said label can be an enzymesuch as horse-radish peroxidase that produces a colorimetric reactionwhen incubated with its substrate such as 3-amino, 9-ethyl-carbazole.

Determination of whether said first protein binds to said second proteinin the presence of the candidate agent can be performed by any suitablemethod depending on the label used. For example, if fluorescent label isused, protein binding can be determined by optical instrument composedof a light source which is preferably a laser and a sensor that detectsthe light emitted from fluorescent label in response to excitation bylight or laser. In the case of calorimetric reaction, such as is thecase when for example horse-radish peroxidase label is used incombination with its substrate such as 3-amino, 9-ethyl-carbazole,protein binding can be determined by change in the absorbance of lightin the wavelength range of interest.

It will be apparent to a person skilled in the art that the method mayalso contain various additional steps, such as incubations and washings.For example, washing may be required after step b) to remove unboundsecond protein.

In one embodiment according to the present invention, the method can beused for a high-throughput screening of inhibitors of protein-proteininteractions by immobilizing a first protein, or a target protein, on a96, 384 well or any equivalent multi-well plate and incubating that witha second protein fused to for example green fluorescent protein, Texasred or luciferase protein. Using a plate reader the light emitted by thefluorescent label is measured and the in a case of interaction the lightsignal is detected from a well. Combining such assay with peptide andsmall molecule compound libraries will allow identification of potentialdrug-like molecules that inhibit protein interaction, detected by lossof fluorescence signal in the chamber.

The present invention is further directed to a method for producing apharmaceutical composition, said method comprising identifying an agentwhich inhibits KIAA1524 and mixing said agent with any suitablepharmaceutically acceptable excipient well known to a person skilled inthe art. In one embodiment, said identification is performed using thescreening and identification method disclosed above.

Furthermore, the present invention is directed to a method of inhibitingKIAA1524 in a human or animal patient in need thereof by administering atherapeutically effective amount of a pharmaceutical compositionproduced according to the present invention.

It is believed that this method can be useful for treating any cancer.However, this method is especially suitable for treating or preventingof cancers located in certain tissues and cancers that would bedifficult or impossible to treat by surgery or radiation. As an exampleof such cancers, can be mentioned squamous cell carcinomas of the headand neck, colon cancer and breast cancer.

It is believed that this method can be also useful for treatinghyperproliferative disease in which KIAA1524 is expressed. An example ofsuch diseases psoriasis, myocardial hypertrophy and benign tumor, suchas adenoma, hamartoma and chondroma.

The method according to this invention can be accomplished either as thesole treating or preventing method, or as an adjuvant therapy, combinedwith other methods such as administration of cytotoxic agents, surgery,radiotherapy, immunotherapy etc.

Agents that may be identified according to various embodiments accordingto the present invention and/or are useful in the methods according tothe present invention include, but are not limited to, oligonucleotides,such as antisense oligonucleotide, siRNA and ribozyme molecule, apeptide, a peptidomimetic, a chemical compound, a small molecule, anantibody raised against said KIAA1524, and an aptamer (anoligonucleotide) affecting the protein conformation of KIAA1524resulting in the inactivation of the same.

According to a preferable embodiment, said agent is an agentdownregulating the expression of KIAA1524.

According to one preferable embodiment, the down regulation of theKIAA1524 is possible, for example, by use of an antisenseoligonucleotide, modified nucleotide, sequence of combination ofdifferent kinds of nucleotides to prevent or modify the KIAA1524synthesis. The antisense oligonucleotide can be a DNA molecule or an RNAmolecule.

Ribozymes cleaving the KIAA1524 mRNA are also included. The ribozymetechnology is described for example in the following publication:Ribozyme technology for cancer gene target identification andvalidation. Li et al., Adv. Cancer Res. 2007; 96:103-43. Also smallinterfering RNA molecules (siRNAs) would be useful. The application ofsiRNA:s has become important in the development of new therapies in thelast years. O Heidenreich presents an overview of pharmaceuticalapplications in the article “Forging therapeutics from small interferingRNAs in European Pharmaceutical Review Issue 1, 2005. The principle hasparticularly been suggested for the treatment of tumors and carcinomas,sarcomas, hypercholesterolemia, neuroblastoma and herpetic stromalkeratitis.

The principle of siRNA is extensively presented in literature. Asexamples can be mentioned the US patent publications 2003/0143732,2003/0148507, 2003/0175950, 2003/0190635, 2004/0019001, 2005/0008617 and2005/0043266. An siRNA duplex molecule comprises an antisense region anda sense strand wherein said antisense strand comprises sequencecomplementary to a target region in an mRNA sequence encoding a certainprotein, and the sense strand comprises sequence complementary to thesaid antisense strand. Thus, the siRNA duplex molecule is assembled fromtwo nucleic acid fragments wherein one fragment comprises the antisensestrand and the second fragment comprises the sense strand of said siRNAmolecule. The sense strand and antisense strand can be covalentlyconnected via a linker molecule, which can be a polynucleotide linker ora non-nucleotide linker. The length of the antisense and sense strandsare typically about 19 to 21 nucleotides each. However, also syntheticdouble-stranded RNA (dsRNA) Dicer substrate duplexes 25-30 nucleotidesin length, which has been recently reported to be more potent thancorresponding conventional 21-mer small interfering RNAs (siRNAs) (Kimet al., 2005), can be used. Typically, the antisense strand and thesense strand both comprise a 3′-terminal overhang of a few, typically 2nucleotides. The 5′-terminal of the antisense is typically a phosphategroup (P). The siRNA duplexes having terminal phosphate groups (P) areeasier to administrate into the cell than a single stranded antisense.In the cell, an active siRNA antisense strand is formed and itrecognizes a target region of the target mRNA. This in turn leads tocleaving of the target RNA by the RISC endonuclease complex(RISC=RNA-induced silencing complex) and also in the synthesis ofadditional RNA by RNA dependent RNA polymerase (RdRP), which canactivate DICER and result in additional siRNA duplex molecules, therebyamplifying the response.

One of the challenges related to small interfering RNAs is theidentification of a potent siRNA for the corresponding mRNA. It shouldbe noted that genes with incomplete complementarity are inadvertentlydownregulated by the siRNA, leading to problems in data interpretationand potential toxicity. This however can be partly addressed bycarefully designing appropriate siRNAs with design algorithms. Thesecomputer programs sieve out given target sequence with a set of rules tofind sequence stretches with low GC content, a lack of internal repeats,an A/U rich 5-end and high local free binding energy which are featuresthat enhance the silencing effect of siRNA.

In order to identify agents useful in the present invention, severaldifferent KIAA1524 siRNAs were designed by using commercial and noncommercial algorithms. To this end, full length cDNA sequence ofKIAA1524 (GenBank accession number NM_(—)020890) was loaded to siRNAalgorithm programs (http://www.mwg-biotech.com/html/ssynthetic_acids/s_sirna_design.shtml) and stand alone program developedby: Wenwu Cuia, Jianchang Ningb, Ulhas P. Naika, Melinda K. Duncana,(OptiRNAi, an RNAi design tool. Computer Methods and Programs inBiomedicine (2004) 75, 67-73). Further, algorithm generated siRNAsequences were then screened trough genome wide DNA sequence alignment(BLAST) (http://www.ncbi.nlm.nih.gov/blast) to eliminate siRNAs whichare not free from off-targeting. In other words, all those siRNAs whichhad even short sequence regions matching with other genes than targetgene (KIAA1524) were considered invaluable for further use. Thisapproach resulted in identification of 5 potential siRNAs depicted inSEQ ID NO:s 2 to 6.

Obtained siRNAs were then transfected to different cell lines and theircapacity to degrade mRNA and further deplete translation of KIAA1524 wasstudied at protein level by measuring the amount of KIAA1524 proteinafter siRNA treatment with KIAA1524 specific antibodies. Those siRNAsequences giving the strongest downregulation of KIAA1524 protein at thelowest concentration used are marked with an asterisk in Table 1.

TABLE 1 Efficiency of siRNAs to downregulate KIAA1524 expression SEQ IDNO Sequence Length Efficiency 2 AACATCAGTGCTTCACTGATCCTT 25 Moderate 3AACTGTGGTTGTGTTTGCACTTT 23 High* 4 GGUUGCAGAUUCUGAAUUATT 21 Moderate 5AAUGCCUUGUCUAGGAUUATT 21 Low 6 ACCAUUGAUAUCCUUAGAATT 21 High*

The present invention thus concerns KIAA1524 siRNAs selected from thegroup consisting of SEQ ID NO:s 2 to 6 and their use as pharmaceuticals.Preferable siRNAs depicted in SEQ ID NO:s 2 and 4, and more preferablesiRNAs depicted in SEQ ID NO:s 3 and 6 are provided. It is obvious thatin some applications said siRNAs may be longer than 21-25 nucleotides ordsRNA dicer substrate duplexes.

The oligonucleotide (such as antisense, siRNA or ribozyme molecule)shall, when used as a pharmaceutical, be introduced in a target cell.The delivery can be accomplished in two principally different ways: 1)exogenous delivery of the oligonucleotide or 2) endogenous transcriptionof a DNA sequence encoding the oligonucleotide, where the DNA sequenceis located in a vector.

Normal, unmodified RNA has low stability under physiological conditionsbecause of its degradation by ribonuclease enzymes present in the livingcell. If the oligonucleotide shall be administered exogenously, it ishighly desirable to modify the molecule according to known methods so asto enhance its stability against chemical and enzymatic degradation.

Modifications of nucleotides to be administered exogenously in vivo areextensively described in the art. Principally, any part of thenucleotide, i.e the ribose sugar, the base and/or internucleotidicphosphodiester strands can be modified. For example, removal of the2′-OH group from the ribose unit to give 2′-deoxyribosenucleotidesresults in improved stability. Prior disclosed are also othermodifications at this group: the replacement of the ribose 2′-OH groupwith alkyl, alkenyl, allyl, alkoxyalkyl, halo, amino, azido orsulfhydryl groups. Also other modifications at the ribose unit can beperformed: locked nucleic acids (LNA) containing methylene linkagesbetween the 2′- and 4′-positions of the ribose can be employed to createhigher intrinsic stability.

Furthermore, the internucleotidic phosphodiester linkage can, forexample, be modified so that one or more oxygen is replaced by sulfur,amino, alkyl or alkoxy groups. Also the base in the nucleotides can bemodified.

Preferably, the oligonucleotide comprises modifications of one or more2′-hydroxyl groups at ribose sugars, and/or modifications in one or moreinternucleotidic phosphodiester linkages, and/or one or more lockednucleic acid (LNA) modification between the 2′- and 4′-position of theribose sugars.

Particularly preferable modifications are, for example, replacement ofone or more of the 2′-OH groups by 2′-deoxy, 2′-O-methyl, 2′-halo, eg.fluoro or 2′-methoxyethyl. Especially preferred are oligonucleotideswhere some of the internucleotide phoshodiester linkages also aremodified, e.g. replaced by phosphorothioate linkages.

It should be stressed that the modifications mentioned above are onlynon-limiting examples.

According to one preferable embodiment, the agent identifiable by thescreening assay or useful in the method according to the presentinvention prevents or inhibits tumor growth and proliferation byinhibiting said KIAA1524 from interacting with PP2A complex or withtranscription factor c-Myc. Alternatively, said agent inhibits KIAA1524growth and proliferation enhancing effects that are not dependent onKIAA1524 interaction with PP2A or c-Myc. Said agent can, for example, bea peptide, peptidomimetic, a small molecule, an antibody raised againstsaid KIAA1524, or an aptamer (an oligonucleotide) affecting the proteinconformation of KIAA1524 resulting in the inactivation of the same.

Results from PR65 co-immunoprecipitation experiments showed thatKIAA1524 interacts both with PR65 and PP2Ac subunits of PP2A proteincomplex. In order to identify the region in KIAA1524 that mediates theprotein-protein interaction, it was studied whether deletion of anyregion in the KIAA1524 protein would abrogate its association with PP2A.To this end, a series of KIAA1524 deletion constructs with each deletioncorresponding to approximately 50-100 amino acids of the protein codingsequence were generated and transiently transfected into HeLa cells. Outof the eleven KIAA1524 deletions, KIAA1524 lacking the amino acidsbetween 461-533 was the only mutant that in repetitive experimentsshowed impaired binding to PR65 as demonstrated byco-immunoprecipitation experiments. Thus, the region ranging from aminoacid 461 to amino acid 533 in SEQ ID NO. 1 is a particularly importanttarget for novel cancer therapeutics.

Thus, according to a particularly preferred embodiment, the agentinactivates the KIAA1524 in the region ranging from aa 461 through 533or any other region on KIAA1524 that participates on interaction betweenPP2A complex and KIAA1524 protein. Said agent can, for example, be apeptide, a peptidomimetic, a small molecule, an antibody raised againstsaid KIAA1524, or an aptamer (an oligonucleotide) affecting the proteinconformation of KIAA1524 resulting in the inactivation of the same.

More specifically, the present invention provides blocking peptidescomprising any stretch of 3 to 60 amino acids, preferably 3 to 30,preferably 6 to 18, preferably 6 to 12, or preferably 9 to 12 aminoacids corresponding to the amino acids of the region aa 461-533 ofKIAA1524 depicted in SEQ ID NO. 1. It is obvious that in someapplications said blocking peptides may be longer that 60 amino acids.Peptides according to the present invention bind to PP2A complexcomponents and inhibit the interaction between KIAA1524 and PP2A therebyeliciting the tumor suppressor activity of PP2A, and on the other hand,inhibiting or blocking KIAA1524. In other embodiments according to thepresent invention, said blocking peptides comprise at least one,preferably 1 to 20, preferably 1 to 10, preferably 2 to 6, preferably 2to 4, or preferably 3 to 4 consecutive sequences selected from the groupconsisting of SEQ ID NO:s 7 to 30 and conservative sequence variantsthereof.

In order to study whether depletion of KIAA1524 affects the functionalinteraction of the PP2A complex with its substrate c-Myc, steady-stateexpression level of c-Myc was studied in HeLa cell extracts aftertransfection with KIAA1524 siRNA. As shown in FIG. 3A, siRNA-induceddepletion of KIAA1524 resulted in down-regulation of c-Myc proteinexpression in HeLa cells, while, as demonstrated in FIG. 3B, c-Myc mRNAexpression was not affected. These results indicated KIAA1524 regulatesc-Myc protein levels post-transcriptionally. To study if the decrease inc-Myc protein levels was caused by protein destabilization, the effectof KIAA1524 depletion on the half-life of endogenous c-Myc protein wasexamined by pulse-chase analysis. As described in more detail in theExperimental Section, KIAA1524 depletion significantly reduced c-Mycprotein stability thus indication that KIAA1524 is an importantregulator of c-Myc stability.

Co-immunoprecipitation of c-Myc and KIAA1524 from cellular extractsdemonstrated a physical association between these proteins (FIG. 3D). Toelucidate the interaction between KIAA1524 and c-Myc further,full-length Flag-tagged recombinant KIAA1524 was produced in insectcells and an in vitro protein-protein interaction assay with recombinantGST-fused amino terminal portion of c-Myc (amino acids 1-262 of thesequence under GenBank accession number NP_(—)002458) was performed.Results indicated that GST-c-Myc 1-262 interacts with KIAA1524, whereasproteolytic degradation fragment of GST-Myc 1-262, corresponding to sizeof GST-Myc 1-120 does not interact with KIAA1524. Therefore, the c-Mycdomain 120-262 is expected to mediate direct binding of c-Myc toKIAA1524. Said c-Myc domain 120-262 contains 143 amino acids andcorresponds to amino acids 1-143 depicted in SEQ ID NO. 31.

The present invention thus provides blocking peptides, which are usefulin inhibiting the interaction of KIAA1524 and c-Myc. In one particularembodiment, blocking peptides comprising any stretch of 3 to 60 aminoacids, preferably 3 to 30, preferably 6 to 18, preferably 6 to 12, orpreferably 9 to 12 amino acids, corresponding to the amino acids 1-143depicted in SEQ ID NO. 31, and inhibiting KIAA1524 are provided. Saidbinding results in inhibition of the interaction between KIAA1524 andc-Myc thereby inhibiting or blocking the KIAA1524. In other embodimentsaccording to the present invention, said blocking peptides comprise atleast one, preferably 1 to 20, preferably 1 to 10, preferably 2 to 6,preferably 2 to 4, or preferably 3 to 4 consecutive sequences selectedfrom the group consisting of SEQ ID NO:s 32 to 79, and conservativesequence variants thereof.

In order to test the blocking ability of the peptides according to thepresent invention, said peptides are administered to cells, such as HeLacancer cells, and, depending on the nature of the peptides, either a)their ability to prevent binding of KIAA1524 to PP2A or b) their abilityto prevent binding of KIAA1524 to c-Myc is detected by any suitablemethod, such as immunoprecipitation analysis of KIAA1524-PP2Ainteraction or by western blot analysis of cellular lysates usingantibodies specific for c-Myc serine 62 phosphorylation. Peptides, whichprevent said binding to a statistically significant extent, areconsidered as blocking peptides.

By the term “conservative sequence variant” it is meant herein variantsarising from amino acid sequence modifications, which do notsignificantly alter the binding properties of the peptides according tothe present invention. Such modifications are apparent to a personskilled in the art, and they include amino acid sequence variantsarising from amino acid substitutions with similar amino acids, as wellas amino acid deletions and additions. With respect to longer peptidesaccording to the present invention, such as peptides comprising morethan 9 amino acids, preferably more than 12, preferably more than 12,preferably more than 18, or preferably more than 30 amino acids, thepresent invention includes those, which have at least 90% identity, orat least 95%, 96%, 97%, 98% or 99% identity to the polypeptidesdescribed above.

When used as a pharmaceutical, blocking peptides according to thepresent invention may be administered by any suitable way, such asintravenous injection, intraperitoneal injection, and intrathecalinjection. In one embodiment, blocking peptides are fused to cellpenetrating peptides (CPPs) known in the art, which deliver the fusionpeptides across the cell membrane. Such fusion peptides may beadministered e.g. by as intravenous injection, intraperitonealinjection, and intrathecal injection. It will be obvious to a personskilled in the art that, as the technology advances, that blockingpeptides and fusion peptides according to present invention may beadministrated by any other suitable way or route of administration.

In addition to the present finding that KIAA1524 is overexpressed inhuman cancer tissues, such as HNSCC, colon cancer and breast cancer, itwas found that the KIAA1524 expression level correlates with the tumorgradus. For example, malignant subtypes of breast cancer expressedstatistically significantly more KIAA1524 than benign subtypes.Furthermore, colon cancers with higher tumor gradus expressedstatistically significantly more KIAA1524 than colon cancers with lowergradus.

This invention concerns also a method for diagnosing cancer orhyperproliferative disease, based on detecting or quantifying the levelof the KIAA1524 protein in a tissue or body fluid by

i) determining the KIAA1524 mRNA expression from said tissue or bodyfluid by RT-PCR, or by a hybridizing technique, or

ii) subjecting the tissue or body fluid expected to contain the proteinKIAA1524 to an antibody recognizing said KIAA1524, and detecting and/orquantifying said antibody, or subjecting said tissue or body fluid toanalysis by proteomics technique.

The hybridizing technique include, for example DNA hybridization andnorthern blot. The detection or quantification of the antibody can beperformed according to standard immunoassay protocols, such aslabel-linked immunosorbent assays, western blot and immunohistochemicalmethods.

This invention concerns also a method for diagnosing cancer orhyperproliferative disease, based on detecting or quantifying themutations or single nucleotide polymorphisms in KIAA1524 gene byhybridizing technique or by DNA or RNA sequencing or by RT-PCR analysisof the RNA or DNA.

The diagnosis can be carried out by KIAA1524 alone or with the same incombination with other proteins or genes.

More specifically, the present invention concerns a method fordetermining the invasiveness of a malignant change in a mammal suspectedto suffer from cancer, said method comprising a) assessing the level ofKIAA1524 expression in a sample, suspected to comprise malignant cells,taken from said mammal, b) comparing the expression level from step a)with the expression level of KIAA1524 in a non-malignant control sample,and c) determining said malignant change as invasive, when theexpression level of KIAA1524 in said sample is significantly higher thanthe expression level of KIAA1524 in a non-malignant control sample.Preferably, the expression level of KIAA1524 in said sample is more than2, preferably more than 3 times higher than in said control sample.

In one embodiment according to the present invention, the above methodis used for determining the gradus of a malignant change in a mammalsuspected to suffer from cancer, such as colon cancer, by determining instep c) the gradus of said malignant change as gradus III or IV, whenthe expression level of KIAA1524 is significantly higher than theexpression level of KIAA1524 in a non-malignant control sample or in anon-invasive gradus I and II sample. Furthermore, the present inventionprovides a method for distinguishing invasive and metastazing gradus IIIand IV from non-invasive and non-metastazing gradus II in cases wheresaid expression level is more than 2 and preferably 3 times higher thanin gradus II or in a non-malignant control sample.

In a further embodiment according to the present invention, the abovemethod is used for distinguishing invasive tumor types of breast cancer,such as invasive ductal carcinoma (IDC), invasive lobular carcinoma(ILC) and IDC with intraductal comedo carcinoma (IDC+ICC), from mucinouscarcinomas in cases where the expression level of KIAA1524 is more than2, preferably more than 3 times higher than in a non-malignant controlsample.

The invention will be illuminated by the following non-restrictiveExperimental Section.

Experimental Section Results Identification of KIAA1524

To identify PP2A interacting proteins from HT-1080 cells we generatedcell clones stably overexpressing TAP-tagged PR65 protein, a scaffoldingsubunit of the PP2A complex (FIG. 1A). TAP purification of cytoplasmicextracts of either mock transfected control or PR65TAP expressing cellsrevealed several proteins that co-purify with PR65TAP but were notpresent in the final eluates from the control cells (FIG. 1B). Severalof these putative PR65 interacting proteins were subsequently identifiedby mass-spectrometric peptide sequencing. Among the proteins identifiedfrom the PR65TAP complex are both the catalytic subunit (PP2Ac) and PP2AB-subunits, thus validating the approach (FIG. 1B). In addition, we wereable to identify KIAA1524 as a novel putative PP2A associated protein.KIAA1524 is a 90 kDA cytoplasmic protein with no previously identifiedcellular function (FIG. 2) (Soo Hoo et al., 2002). Results presentedbelow, identify KIAA1524 as an endogenous inhibitor of PP2A specificallyup-regulated in cancer.

Results from PR65 co-immunoprecipitation analysis show that endogenousKIAA1524 interacts with endogenous PR65 and PP2Ac (FIG. 1C). In order toidentify the region in KIAA1524 that mediates the protein-proteininteraction between KIAA1524 and PP2A complex, we have constructed aseries of cDNA constructs coding for KIAA1524 deletion mutants. To thisend, a series of Flag-tagged KIAA1524 deletion constructs weretransiently transfected into HeLa cells for 48 h, followed byimmunoprecipitation with anti-Flag antibody. Interaction between theKIAA1524 mutants and the PP2A complex was assessed by Western blotanalysis of the PR65 subunit. Immunoprecipitation of Flag-KIAA1524wild-type (KIAA1524 wt) (FIG. 2A) construct revealed a clear interactionwith endogenous PR65 protein (FIGS. 1D and 3A). Interaction occurred inboth low (150 mM NaCl) or moderate (300 mM NaCl) stringency conditions.However, a mutant of KIAA1524, lacking the amino acids between 461-533(KIAA11524mut)(FIG. 2A), displayed greatly reduced interaction with PR65(FIGS. 1D and 3A). KIAA1524mut was the only mutant out of elevendeletion mutants that demonstrated impaired binding to PR65 in eitherstringency condition.

Next we studied the co-localization of PP2Ac and KIAA1524 from cellsco-transfected with either KIAA1524 wt or KIAA1524mut constructstogether with HA-PP2Ac. As shown in FIG. 1E, confocal image analysis oftransfected cells revealed co-localization with PP2Ac and KIAA1524 wt,whereas no co-localization was observed with PP2Ac and KIAA11524mut(FIG. 1E). In order to study the role of KIAA1524 in the regulation ofPP2A function, we have inhibited KIAA1524 expression in HeLa cells usingshort interfering RNA (siRNA) oligos. Importantly, depletion of KIAA1524expression by siRNA stimulated PP2A phosphatase activity as measuredfrom the PP2Ac immunoprecipitates (FIG. 1F). Taken together theseresults demonstrate that KIAA1524 interacts with and inhibits thecatalytic activity of the PP2A complex in cultured cells. Moreover, thedata show that the interaction is stable at moderate ionic strength andthat amino acids 461-533 of KIAA1524 contain the PP2A interaction domainof KIAA1524.

KIAA1524 Promotes c-Myc Protein Stability

To probe for the uncharacterized cellular functions of KIAA1524,genome-wide gene expression profiles of scrambled and KIAA1524 siRNAtransfected HeLa cells were compared after 72 h. Remarkably, only aminor fraction of genes (76 out of 26091) included in the Sentrix®Human-6 Expression BeadChip (Illumina Inc.), showed a reproducible andstatistically significant (p<0.05, data not shown) difference in theirexpression levels between scrambled and KIAA1524 siRNA transfected cells(FIG. 1F). PP2A activity has been shown to regulate the activity of twotransformation relevant transcription factors, p53 and c-Myc. Tocharacterize these two transcription factors in relation to thetranscriptional profile of KIAA1524 depleted cells, the list of 76 genesaffected by KIAA1524 depletion was compared to target gene databasespublished for p53 and c-Myc. Based on a p53 target gene database(http://p53.bii.a-star.edu.sg/aboutp53/targetgene/index.php), only 1/76genes affected by KIAA1524 depletion has been published to harbor a p53binding site in its promoter or to be transcriptionally regulated byp53. However, when compared with a c-Myc target gene database(http://www.myc-cancer-gene.org/site/mycTargetDB.asp), 16% (12/76) ofgenes affected by KIAA1524 depletion were found to bind c-Myc in theirpromoter region. These findings, together with the published role ofPP2A regulation of c-Myc protein stability, suggest that KIAA1524 mayregulate c-Myc function.

As described above, viral small-t antigen leads to stabilization of thec-Myc protein by protecting c-Myc serine 62 from PP2A-mediateddephosphorylation. In order to study if depletion of KIAA1524 regulatesc-Myc expression, we examined c-Myc steady-state protein levels bywestern blotting from cells transfected with KIAA1524 or scrambled(Scr.) siRNA. KIAA1524 siRNA treatment resulted in clear downregulationof c-Myc protein expression, whereas c-Myc mRNA expression levels werenot altered in the same samples (FIGS. 4A and 48). This implies thatKIAA1524 regulates c-Myc protein levels post-transcriptionally. Indeed,the analysis of the effect of KIAA1524 depletion on the half-life ofendogenous c-Myc protein revealed that whereas in scrambled siRNAtransfected cells approximately 40% of c-Myc protein was present incells 1 h after cycloheximide treatment (100 mg/ml), KIAA1524 depletionsignificantly reduced c-Myc protein stability.

KIAA1524 Inhibits c-Myc-Associated PP2A Activity

The results above demonstrate that KIAA1524 interacts with the PP2Acomplex and promotes c-Myc stability. To study whether KIAA1524 indeedinhibits c-Myc-associated PP2A activity, c-Myc immunoprecipitates fromscrambled and KIAA1524 siRNA transfected cells were subjected to an invitro PP2A assay using 6,8-difluoro-4-methylumbelliferyl phosphate asthe substrate (Pastula et al., 2003) Importantly, KIAA1524 depletion bysiRNA markedly increased PP2A activity in c-Myc immunoprecipitates (FIG.4C). Interestingly, analysis of immunoprecipitates further revealed thatPP2Ac and c-Myc are constitutively in complex with each other and thatdepletion of KIAA1524 has no effect on c-Myc-PP2Ac interaction (FIG.4D). Together, these results demonstrate that KIAA1524 inhibits PP2Aactivity in the c-Myc-PP2A complex and prevents proteolytic degradationof c-Myc.

Co-immunoprecipitation of c-Myc and KIAA1524 from cellular extractsdemonstrates a physical association between these proteins (FIG. 4D),but does not reveal whether the interaction between KIAA1524 and c-Mycis direct. To further characterize KIAA1524's interaction with c-Myc,purified Flag-KIAA1524 protein was used in an in vitro protein-proteininteraction assay with recombinant GST-fused aminoterminal portion ofc-Myc (aa. 1-262). We found that Flag-antibody resinco-immunoprecipitated recombinant Flag-KIAA1524 and GST-Myc, whereasFlag-KIAA1524 did not co-immunoprecipitate with GST alone (FIG. 5A),demonstrating that KIAA1524 directly binds to the c-Myc aminoterminus.Interestingly, KIAA1524 did not co-immunoprecipitate with GST-c-Mycdeletion corresponding to aminoacids 1-120 (FIG. 5A). These resultsindicate that KIAA1524 interaction domain in c-Myc is between aminoacids120-262 on c-Myc (FIG. 5B).

Together these experiments provide solid biochemical evidence thatKIAA1524 inhibits c-Myc-associated PP2A activity. Moreover, directbinding of KIAA1524 to c-Myc aminoterminus provides the most feasibleexplanation for the observed selectivity of KIAA1524 towardsc-Myc-associated PP2A activity.

KIAA1524 is Required for Tumor Growth

In order to study the role of KIAA1524 in regulation of cancer cellbehavior, we transfected HeLa cells with KIAA1524 siRNA and studied cellproliferation in both cell culture and in in vivo mouse model. The roleof KIAA1524 in regulating cell proliferation was next analyzed bythymidine incorporation assay in HeLa cells. Compared to scrambled siRNAtransfected cells, KIAA1524 depletion resulted in significant inhibitionof cell proliferation 72 h after transfection (FIG. 6A). However,KIAA1524 siRNA transfection did not induce a sub-G1 fraction in FACSanalysis of the cellular DNA content (FIG. 6B), nor did it inducecleavage of the PARP protein (FIG. 6C), demonstrating that KIAA1524depletion does not induce programmed cell death.

To assess the contribution of KIAA1524 on the tumorigenic potential ofHeLa cells, the effects of KIAA1524 depletion on the ability of thesecells to form dense foci on a monolayer, as well as their ability togrow in an anchorage-independent manner was analyzed. For this purpose,we first studied the efficiency of KIAA1524 depletion by a singletransfection of siRNA 10 days after transfection and found thatapproximately 50% of KIAA1524 protein expression was still reduced after10 days. KIAA1524 depletion abrogated HeLa cell foci formation 10 daysafter transfection (FIG. 6D) and also clearly inhibited HeLa cellanchorage independent growth on agar as measured 10 days after platingof the cells on agar (FIG. 6E).

To assess the tumorigenic role of KIAA1524 in vivo, HeLa cellstransfected with either KIAA1524 or scrambled siRNA for 72 h weresubcutaneously injected into athymic mice and tumor growth was monitoredby measuring the size of palpable tumors. Importantly, depletion ofKIAA1524 by siRNA reduced the overall tumor size (FIG. 6F), and resultedin a significant inhibition of tumor weight at day 27 (FIG. 6G).

Taken together, these data demonstrate that KIAA1524 expression is anovel mechanism of maintenance for the transformed cellular phenotypeand that KIAA1524 promotes in vivo tumor growth.

KIAA1524 is Overexpressed in Human Malignancies

Results presented above provide evidence that KIAA1524 inhibitsPP2A-mediated c-Myc degradation and promotes cancer cell growth andproliferation. Based on these characteristics, KIAA1524 could be a noveldrug target for cancer therapeutics. In order to fulfill theexpectations for a protein targeted in cancer therapies, KIAA1524 shouldpreferably be overexpressed in human cancer tissues. According to ourquantitative RT-PCR analysis, KIAA1524 mRNA was expressed at very lowlevels (<1% of βactin mRNA expression levels) in the majority of the 21non-malignant samples, with the exception of bone marrow, prostate,testis, cerebellum and brain (FIG. 7A). To compare the protein levels ofKIAA1524 between non-malignant and malignant cells, whole cell lysatesof different cell types were immunoblotted for KIAA1524. Consistent withFIG. 7A, very low levels of KIAA1524 protein was detected in humanepidermal keratinocytes (HEK), non-tumorigenic MEFs and immortalizedNIH3T3 mouse fibroblasts. However, KIAA1524 protein was expressed athigh levels in both HeLa cells and in HT-1080 fibrosarcoma cells,indicating that KIAA1524 expression may correlate with the tumorigenicpotential of cells.

Furthermore, KIAA1524 expression levels in human squamous cellcarcinomas of the head and neck (HNSCC) were analyzed. Real-time PCRanalysis of KIAA1524 mRNA showed statistically significantoverexpression of KIAA1524 in 36 HNSCC cell lines as compared to normalhuman epidermal keratinocytes used as a control (FIG. 7B). KIAA1524 mRNAwas also overexpressed in HNSCC tumor biopsies as compared to benigncontrol tissues from the same region of the body (FIG. 7C). Importantly,immunohistochemical staining of HNSCC samples also confirmed higherexpression of KIAA1524 in tumor cells as compared to surrounding stromalcells (FIG. 7D).

Finally, in order to study if KIAA1524 regulates c-Myc protein levelsalso in primary cancer cell lines derived from HNSCCs, three differentHNSCC cell lines were transfected with KIAA1524 siRNA and analyzed forc-Myc expression by western blotting. As shown in FIG. 7E, KIAA1524depletion resulted in clear downregulation of c-Myc protein levels inall cell lines examined. Importantly, analysis of c-Mycimmunoprecipitates revealed that KIAA1524 depletion also increased c-Mycassociated PP2A phosphatase activity in HNSCC cell lines.

To examine the role of KIAA1524 in the regulation of HNSCC cellproliferation, UT-SCC-7 and UT-SCC-9 cell lines were subjected toKIAA1524 siRNA transfection and dense foci formation of these cell lineswas monitored for 10 days. In both cell lines, depletion of KIAA1524resulted in a significant reduction in foci formation. Importantly,KIAA1524 depletion also significantly reduced anchorage independentgrowth of both UT-SCC-7 and UT-SCC-9 cells in soft agar 21 days aftertransfection. Consistent with the demonstrated specificity of KIAA1524siRNAs in HeLa cells, two independent KIAA1524 siRNAs yielded a similarinhibition in soft agar growth of UT-SCC-9 cells.

Finally, to assess the role of KIAA1524 for in vivo tumor growth ofUT-SCC cells, both UT-SCC-7 and UT-SCC-9 cells transfected with KIAA1524or scrambled siRNAs were injected into the back of SCID mice. Consistentwith all the other data presented in this work indicating the importanceof KIAA1524 for malignant cell growth and tumor progression, only 3 outof 5 and 2 out of 6 mice injected with KIAA1524 siRNA transfectedUT-SCC-7 and UT-SCC-9 cells, respectively, developed palpable tumors atday 65 when the experiment was terminated. Moreover, KIAA1524 siRNAreduced the average size of tumors with both of the UT-SCC cells, ascompared to scrambled siRNA transfected cells.

To confirm that the above results are restricted to HNSCC, KIAA1524expression was analyzed from 43 human colon cancer samples and controlsamples from normal colon by RT-PCR. In accordance with the HNSCC data,KIAA1524 mRNA was significantly overexpressed in human colon cancertissues as compared to control tissues (FIG. 8A).

Furthermore, comparison of KIAA1524 mRNA expression to colon cancertumor gradus revealed that KIAA1524 expression is significantly higherin invasive gradus III and gradus IV tumors as compared to non-invasivegradus II tumors or control colon tissue (FIG. 8B). The tumor gradus ofthe tissue samples used for the analysis have previously been determinedby standard pathological criteria.

In order to study if KIAA1524 is over-expressed, in addition to HNSCCand colon cancer, also in breast cancer samples, KIAA1524 expression wasevaluated in 159 previously characterized human mammary tumors andnormal breast samples (Come et al., 2006). Importantly, it was foundthat KIAA1524 is significantly over-expressed in human mammary tumorswhen compared to normal tissue (FIG. 9A). When comparing KIAA1524expression between breast cancer sub-types, over-expression of KIAA1524was found in all three invasive mammary cancer sub-types, invasiveductal carcinoma (IDC), invasive lobular carcinoma (ILC) and IDC withintraductal comedo carcinoma (IDC+ICC) (FIG. 9B). As a control, mucinouscarcinomas, that are mammary tumors with good prognosis, presentedsimilar KIAA1524 mRNA expression than normal breast, statistically lowerthan invasive tumors (FIG. 9B).

Taken together, results of this demonstrate that KIAA1524 promotes tumorgrowth and cancer cell proliferation and strongly indicate thatsuppression of c-Myc associated PP2A activity is at least one of themolecular mechanisms by which KIAA1524 exerts its cellular effects.

Materials and Methods Antibodies

Rabbit polyclonal antibody for KIAA1524 has been published (Soo Hoo etal., 2002)(generously provided by Dr. Chan, University of Florida).Antibodies for PP2Ac, PR65, Flag, HA, GST, PARP, c-Myc and Actin wereobtained from Santa Cruz biotechnologies inc.

Plasmid Constructs

PR65TAP-tag vectors PR65α was PCR amplified from pRC/CMV.HA PR65a (akind gift from Dr. Brian A. Hemmings, Friedrich Miescher-Institut,Basel, Switzerland),) and cloned into the TAP vector, JW16 (Westermarcket al., 2002), using XhoI and BamHI sites. Flag-KIAA1524 wt constructswas constructed with PCR from published KIAA1524 full-length cDNA (SooHoo et al., 2002) (generously provided by Dr. Chan, University ofFlorida). Flag-KIAA1524mut cDNA construct was constructed with PCR fromplasmid Flag-KIAA1524 wt with PCR using oligos:5-Ttaatagagaaacttcagtctggaatg (SEQ ID NO. 80) and5-Gtggtaaaggatcagatttgtgatgtgaga (SEQ ID NO. 81). All clones werethereafter verified by DNA sequencing.

Patient Samples

After informed consent, tumor samples were collected from surgicallyremoved tumors from HNSCC between years 1990-2002 in Turku UniversityCentral Hospital. Normal samples were collected from patients undergoinguvulo-palato-pharyngoplasty for a HNSCC study. Samples were collectedfrom both genders ranging in age from 29-87 years old.

Expression of KIAA1524 in colon cancer and normal colon tissue wasexamined by using TissueScan Real-Time Colon Cancer (HCRT101) cDNA panel(Origene), containing 43 samples from colon cancer (both genders rangingin age from 31-93 years) and 5 samples of normal colon tissues (bothgenders ranging in age from 37-91 years). Total RNA extracted from 21different normal human tissues was obtained from BD Biosciences (PaloAlto, Calif.) and was a generous gift from Prof. Klaus Elenius,University of Turku, Finland. Samples consisted either of RNAextractions from single patients (cerebellum, brain, heart, liver, andlung) or of pooled RNA extractions from 2 to 84 patients (adrenal gland,bone marrow, kidney, placenta, prostate, salivary gland, skeletalmuscle, spleen, thymus, thyroid gland, trachea, uterus, colon, smallintestine, and mammary gland).

Expression of KIAA1524 in breast cancer and normal mammary tissue wasexamined by using previously described tissue samples (Come et al.,2006).

Cell Cultures

Human SCC cell lines were established from primary tumors (UT-SCC-8),recurrent tumors, or metastasis (UT-SCC-7, UT-SCC-9) of head and neckSCCs. SCC cells were cultured in DMEM supplemented with 6 nmol/lglutamine, nonessential amino acids, 100 U/ml penicillin, 100 mgstreptomycin, and 10% fetal calf serum (FCS). Normal human epidermalkeratinocytes were cultured in Keratinocyte Basal Medium 2 (KBM®-2)supplemented with SingleQuots® (Cambrex Bioscience; Walkersville, Md.,USA). All other cells lines, including HT-1080, HeLa and HK293 werecultured in DMEM supplemented with 100 U/ml penicillin, 100 mgstreptomycin, and 10% FCS.

Transient Transfections and siRNA Treatment

Subconfluent cells were transiently transfected using FuGene 6Transfection reagent (Roche) according to the manufacturer'sinstructions. For siRNA treatments, cells were grown to 80% confluenceand medium was replaced with DMEM without supplements. Double-strandedRNA oligonucleotides (scrambled: 5′-UAACAAUGAGAGCACGGCTT-3′ (SEQ ID NO.82) and 5′-CCUACAUCCCGAUCGAUGAUGTT-3′ (SEQ ID NO 83); KIAA1524:5′-CUGUGGUUGUGUUUGCACUTT-3′ (SEQ ID NO. 84) and5′-ACCAUUGAUAUCCUUAGAATT-3′ (SEQ ID NO 6); IBA) were prepared withOligofectamine™ reagent (Invitrogen) and added to the cells. After 4-6 hincubation, the medium was equilibrated to 10% FCS and the siRNAtreatment was extended for the appropriate length of time.

RNA Isolation and cDNA Synthesis

Total RNA was extracted from cultured cells using TRIzol reagent(Invitrogen) according to the manufacturer's protocol. From clinicaltumor samples, RNA was extracted using acid-guanidiumthiocyanate-phenol-chloroform method. To eliminate possiblecontaminating DNA, RNA samples were treated with 5 units of DNase I(Roche). cDNA was synthesized in a reaction containing 1 μg of total RNAas a template, 0.5 μg random hexamers and 200 units of Moloney murineleukemia virus RNase H minus reverse transcriptase (both from Promega;Madison, Wis., USA), in a total volume of 25 μl according to themanufacturer's protocol.

Immunofluorescence

24 hours after transfection with HA-PP2Ac and Flag-KIAA1524 cDNAconstructs, HeLa cells cultured on glass coverslips were permeabilizedand fixed for 10 minutes in PTEMF (100 mM Pipes (pH 6.8), 10 mM EGTA, 1mM MgCl2, 0.2% Triton X-100, and 4% formaldehyde). After three washeswith PBS nonspecific antibody binding was blocked for 30 minutes with 3%BSA in PBS. Coverslips were thereafter incubated with HA and Flag-tagspecific antibodies for 1 h in RT in 2% BSA in PBS. After three washes,bound antibodies were visualized by incubation with Cy3 and Cy2 and-conjugated secondary antibody (Jackson ImmunoResearch) for 1 hour atroom temperature. After three washes with PBS, the coverslips weremounted in 50% glycerol, PBS, and 2% w/v DABCO (Sigma-Aldrich). Foranalysis of co-localization, images were aquired using a confocal laserscanning microscope (LSM 510, Carl Zeiss Inc.).

Immunoprecipitations and Phosphatase Assays

Protein G-Sepharose beads were agitated on a tumbler for 2 h at 4 C withPR65, PP2Ac, c-Myc antibodies or with control pre-immune serum in 20 mMHEPES-KOH pH 7.5, 300 mM NaCl, 0.25 mM EGTA, 1.5 mM MgCl2, 0.25% NP-40,protease inhibitors (Roche), 10 mM b-glyserolphosphate and 0.5 mM DTT.Immunoprecipitation of Flag-KIAA1524 mutants was performed by usingFlag-antibody resin (M3, Sigma). Thereafter, sedimented beads were mixedwith cellular lysates and incubated for over-night in 4 C. Thereafterthe sedimented beads were washed four times with 50 mM Tris-HCl pH 7.4,150 mM NaCl, 0.3% NP-40 and 0.5 mM DTT, and bound proteins were analyzedby western blotting using True-blot secondary antibodies. Forphsophatase assays, immunocomplexes were retained on beads and equalamounts of beads were added to phosphatase assays using the ProteinSerine/Threonine Phosphatase Assay kit with6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) substrate(Molecular Probes, Eugene, Oreg. USA) according to the manufacturer'sinstructions.

To study direct interaction between KIAA1524 and c-Myc, Flag-KIAA1524was baculovirally expressed in insect cells and affinity purified tohomogeneity by Flag-antibody resin (M3, Sigma), as described previously(Nordlund et al., 2005). For interaction assays Flag-KIAA1524 proteinwas eluated from Flag-Ab beads by using low Ph conditions. The purityand quantity of this KIAA1524 protein was analyzed in each step bySDS-page gel and coomassie staining. No other contaminating proteinswere identified in the KIAA1524 preparates. GST protein was bacteriallyproduced and affinity purified according standard protocols andGST-Myc1-262 was purchased from Santa Cruz Inc. Pull-down assay wasperformed by incubating 1 μg of Flag-KIAA1524 immobilized on anti-FlagM3 resin (Sigma) with molar equivalent amounts of soluble GST-c-Myc andGST proteins in 0.5 ml of buffer containing 20 mM Tris (pH 7.4), 0.2 mMEDTA, 0.1 M NaCl, 0.5 mM DTT and Complete protease inhibitors (Roche).Absence of insect PP2A in KIAA1524 protein preparate was controlled byphosphatase assay. Anti-Flag M3 resin was used as a control resin in allexperiments. Bound proteins were washed in binding buffer supplementedwith 0.2% NP-40 and subsequently boiled in sample buffer, resolved inSDS-PAGE and immunoblotted with GST and KIAA1524 antibodies.

Quantitative Reverse Transcription-PCR Analysis

Quantitative real-time reverse transcription-PCR (RT-PCR) analysis ofcDNA samples was performed with specific primers and fluorescent probesdesigned using Primer Express software (PE Biosystems) to specificallyquantitate the levels of KIAA1524 and β-actin mRNA. The sequences of theprimers and probes are shown below:

KIAA1524: (SEQ ID NO. Probe: att gct cag cat cgc tgt caa aga act ca SEQID NO. 86) Forward: aag ctc tag ccc ttg cac agg (SEQ ID NO. 87) Reverse:gtc cgt gcc tct gtt tca gc β-actin: (SEQ ID NO. 88) Probe: atg ccc tccccc atg cca tcc tgc gt (SEQ ID NO. 89) Forward: tca ccc aca ctg tgc ccatct acg c (SEQ ID NO. 90) Reverse: cag cgg aac cgc tca ttg cca atg g85)

PCR was carried out in a solution containing 300 nM of primers(Medprobe), 200 nM of 5′ 6-FAM (PE Biosystems), 12.5 μl of TaqManuniversal PCR Master Mix (PE Biosystems), and 0.5 μl of template cDNA ina final volume of 25 μl. Thermal cycling was performed with ABI PRISM7700 Sequence Detector (PE Biosystems). Cycling was initiated with 2 minat 50° C. and 10 min at 95° C., followed by 40 cycles of 15 s at 95° C.and 1 min at 60° C. Accumulation of the specific PCR products wasdetected real-time as an increase in fluorescence. Observed fluorescencewas plotted against cycle number to generate amplification plots and todetermine CT values, i.e. the cycle numbers at which the fluorescencesignal exceeded a CT value of 0.05 relative fluorescence units. Eachdetermination of a CT value was done in duplicate and normalized withthe CT values of simultaneous duplicate measurements of β-actinexpression from the same samples. The range between two parallel CTvalues was <5% of the mean in all of the measurements. Relativeexpression of the gene analyzed (target gene) was estimated using theformula: relative expression=2−ΔCT, where ΔCT=CT(targetgene)−CT(β-actin). The quantity of mRNAs was expressed as percentage ofthe quantity of β-actin mRNA after multiplying relative target geneexpression by a factor of 100.

Soft Agar Growth, Foci Formation and Tumor Formation in Mouse

For foci formation assay and anchorage-independent growth in soft agar,HeLa-cells were trypsinated and seeded to 4×105 cells at 10 cm plates 48hours after siRNA treatment. Soft-agar assays were performed in mediumcontaining 10% FBS as described in the literature. After 9 daysincubation cultures were photographed double-blindly. The numbers ofviable colony formatting cells were measured from microscopy images (×5magnification). The number and size of colonies from each image of viewwere analyzed using ImageJ 1.33u software from NIH(http://rsb.info.nih.gov/ij/). Anchorage independent colonies wereclassified according to number between 200-10,000 pixels.

For foci formation assays in MEFs, 200 or 500 cells of the retrovirallytransduced MEFs were plated with 8.3×105 NIH3T3 cells as feeders perwell in a 6 well plate. Cells were grown for 1-2 weeks. Methanol-fixedcells were stained with Giemsa. Foci numbers were calculated per 100infected cells.

For the mouse experiments, 3×10⁶ transfected cells were injectedsubcutaneously to the flank of immunocompromised mouse. Tumor formationwas evaluated thereafter every third day by palpation, and the size ofthe palpable tumors was measured by precision instrument. The experimentwas terminated at the day 28. All experiments with mice were performedaccording to institutional animal care guidelines and with thepermission of the animal test review board of the University of Turku,Finland.

Western Blot Analysis

Following protein separation by SDS-PAGE gel electrophoresis theproteins were transferred to Immobilo-P membrane (Millipore; Billerica,Mass., USA). After incubation with primary and secondary antibodies,immunoblotted proteins were visualized by enhanced chemiluminescence(ECL; Amersham Biosciences).

Immunohistochemistry

Immunostaining of paraffin-embedded tumor sections and control tissuesections for KIAA1524 was performed using 1:100 dilution of p90 antibody(Soo Hoo, et al., 2002) in PBS. Immunostaining was done withavidin-biotin-peroxidase complex technique (VectaStain; Vector Labs;Burlingame, Calif., USA) in combination with diaminobenzidine (DAB), andcounterstained with hematoxylin.

Preparation of Lysates for Tandem Affinity Purification

HT-1080 cells stably expressing PR65TAP protein were washed withphosphate buffered saline (PBS) and resuspended in Buffer A (10 mM HepespH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, CompleteProtease Inhibitor, 20 mM b-glycerolphosphate, 25 mM NaF, 0.5 mM PMSF,and 0.5 mM DTT). Cells were incubated on ice, vortexed and thencentrifuged at 3900 rpm for 3 min to obtain the cytoplasmic lysate.Extracts were then adjusted to contain similar levels of NaCl and NP-40as IgG Binding Buffer (IBB; 10 mM Tris HCl pH 8.0, 150 mM NaCl, 0.2%NP-40, 0.5 mM DTT). Adjusted extracts were then loaded onto Poly-Prep®chromatography columns (BioRad) containing IgG sepharose 4 Fast Flow(Amersham) washed with IBB. Extracts were incubated for 4 h at 4° C.followed by 3×10 ml washes of IBB. The beads were then incubated in TEVcleavage buffer (TCB; 10 mM Tris HCl pH 8.0, 150 mM NaCl, 0.3% NP-40,0.5 mM EDTA, and 0.5 mM DTT) and recombinant tobacco etch virus protease(TEV) overnight at 4° C. Calmodulin beads (Startagene) are washed inPoly-Prep® chromatography columns with Calmodulin Binding Buffer (CBB;10 mM b-mercaptoethanol, 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mMMg-acetate, 1 mM imidazole, 2 mM CaCl2, and 0.2% NP-40). The TEV eluatewas then recovered from the column and adjusted for binding tocalmodulin beads (4 ml of 1 M CaCl2 and 3 ml of CBB for every 1 ml ofeluate). The adjusted eluate is incubated for 2 h at 4° C. and thenwashed 3×10 ml with CBB. The proteins bound to the calmodulin beads arethen recovered either with Calmodulin Elution Buffer (CEB; 10 mMb-mercaptoethanol, 10 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM Mg-acetate,1 mM imidazole, 5 mM EGTA, and 0.2% NP-40) or boiled in SDS loadingbuffer.

Mass Spectrometric Protein Identification

The protein bands of interest were excised from the gel, reduced,alkylated, and digested overnight with trypsin as described previously.Extracted peptides were characterized by LC-MS/MS on a hybrid linearion-trap instrument (Q-Trap, Applied Biosystems, Framingham, Mass.,USA), coupled to a nanoflow HPLC system (LC Packings, San Francisco,Calif., USA). The resulting peptide fragment spectra were searchedagainst a comprehensive non-redundant protein database, using Mascot. Aminimum of three matching tryptic peptides was required to identify eachprotein, and correct fragment ion assignment was guaranteed by manualinspection, if necessary.

Gene Expression Analysis

Total RNA extracted from HeLa cells 72 h after transfection with eitherscrambled or KIAA1524 siRNA was analysed for genome-wide gene expressionprofiles by Sentrix® Human-6 Expression BeadChip array (Illumina Inc.).cDNA amplification, labeling and hybridization were done according themanufacturers instruction and standard procedures at the Finnish DNAMicroarray Centre (Centre for Biotechnology, University of Turku and ÅboAkademi University, Turku, Finland). The data obtained from array wasanalyzed by Bioinformatics core facility personal at the Centre forBiotechnology, University of Turku and Åbo Akademi University, Turku,Finland. The list 76 genes which expression was significantly altered inresponse to KIAA1524 depletion was filtered following criteria of atleast log 0.5 change in the expression levels in both of the twoexperiments, as compared to scrambled siRNA transfected cells.

Statistical Methods

For FIGS. 6G, 8A, 8B, 9A and 9B the statistical significance wasdetermined by Mann-Whitney U test and for all the other experimentsdisplaying statistical analysis it was performed by Student's t-test.

It will be appreciated that the methods of the present invention can beincorporated in the form of a variety of embodiments, only a few ofwhich are disclosed herein. It will be apparent for the expert skilledin the field that other embodiments exist and do not depart from thespirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

REFERENCES

-   Come, C., Magnino, F., Bibeau, F., De Santa Barbara, P., Becker, F.    K., Theillet, C. and Savagner, P. (2006) Clinical Cancer Research,    12, 5395-5402.-   Janssens, V. and Goris, J. (2001) Protein phosphatase 2A: a highly    regulated family of serine/threonine phosphatases implicated in cell    growth and signalling. Biochem J, 353, 417-439.-   Kim D. H, Behlke M. A, Rose S. D, Chang M. S, Choi S and    Rossi J. J. (2005) Synthetic dsRNA Dicer substrates enhance RNAi    potency and efficacy. Nat Biotechol, 23, 222-226.-   Nordlund, H. R., Laitinen, O. H., Uotila, S. T., Kulmala, M.,    Kalkkinen, N., and Kulomaa, M. S. (2005). Production of Hev b5 as a    fluorescent biotin-binding tripartite fusion protein in insect    cells. Biochem Biophys Res Commun 336, 232-238.-   Pastula, C., Johnson, I., Beechem, J. M., and Patton, W. F. (2003).    Development of fluorescence-based selective assays for    serine/threonine and tyrosine phosphatases. Comb Chem High    Throughput Screen 6, 341-346.-   Soo Hoo, L., Zhang, J. Y. and Chan, E. K. (2002) Cloning and    characterization of a novel 90 kDa ‘companion’ auto-antigen of p62    overexpressed in cancer. Oncogene, 21, 5006-5015.-   Westermarck, J., Weiss, C., Saffrich, R., Kast, J., Musti, A. M.,    Wessely, M., Ansorge, W., Seraphin, B., Wilm, M., Valdez, B. C. and    Bohmann, D. (2002) The DEXD/H-box RNA helicase RHII/Gu is a    co-factor for c-Jun-activated transcription. EMBO J, 21, 451-460.-   Yeh, E., Cunningham, M., Arnold, H., Chasse, D., Monteith, T.,    Ivaldi, G., Hahn, W. C., Stukenberg, P. T., Shenolikar, S., Uchida,    T., Counter, C. M., Nevins, J. R., Means, A. R. and Sears, R. (2004)    A signalling pathway controlling c-Myc degradation that impacts    oncogenic transformation of human cells. Nat Cell Biol, 6, 308-318.-   Zhao, J. J., Roberts, T. M. and Hahn, W. C. (2004) Functional    genetics and experimental models of human cancer. Trends Mol Med,    10, 344-350.

1. A method for screening and identifying a therapeutic agent, whichinhibits KIAA1524, said method comprising the steps of: a) providing afirst protein immobilized in a reaction chamber, b) adding a candidateagent and a labeled second protein to said chamber concomitantly orsubsequently in any order, c) determining whether said first proteinbinds to said second protein, and d) identifying said candidate agent asa therapeutic agent inhibiting KIAA1524 when the determination in stepc) is negative, wherein said first protein is KIAA1524 and said secondprotein is selected from the group consisting of PP2A, subunits thereof,and c-Myc, or vice versa.
 2. The method according to claim 1, whereinsaid second protein is labeled with a label selected from the groupconsisting of fluorescent labels, luminescent labels and calorimetriclabels.
 3. The method according to claim 1, wherein step c) is performedby a plate reader.
 4. Small interfering RNA comprising a nucleotidesequence selected from the group consisting of SEQ ID NO:s 2 to 6, andinhibiting KIAA1524.
 5. A peptide comprising at least one amino acidsequence selected from the group consisting of SEQ ID NO:s 7 to 30, 32to 79, and conservative sequence variants thereof, and inhibitingKIAA1524.
 6. The peptide according to claim 5, wherein said peptidecomprises 1 to 20 consecutive sequences selected from the groupconsisting of SEQ ID NO:s 32 to 79 and conservative sequence variantsthereof.
 7. The peptide according to claim 5, wherein said peptidecomprises 1 to 20 consecutive sequences selected from the groupconsisting of SEQ ID NO:s 7 to 30 and conservative sequence variantsthereof.
 8. A method for producing a pharmaceutical composition, saidmethod comprising identifying an agent which inhibits KIAA1524 andmixing said agent with any suitable pharmaceutically acceptableexcipient.
 9. The method according to claim 8, wherein saididentification is performed by a method for screening and identifying atherapeutic agent which inhibits KIAA1524, said method comprising thesteps: a) providing a first protein immobilized in a reaction chamber,b) adding a candidate agent and a labeled second protein to said chamberconcomitantly or subsequently in any order, c) determining whether saidfirst protein binds to said second protein, and d) identifying saidcandidate agent as a therapeutic agent inhibiting KIAA1524 when thedetermination in step c) is negative, wherein said first protein isKIAA1524 and said second protein is selected from the group consistingof PP2A, subunits thereof, and c-Myc, or vice versa.
 10. Apharmaceutical composition produced according to claim
 8. 11. A methodof inhibiting KIAA1524 in a human or animal patient in need thereof byadministering a therapeutically effective amount of a pharmaceuticalcomposition according to claim
 10. 12. The method according to claim 11,wherein said patient suffers from a disease selected from the groupconsisting of cancer and other hyperproliferative diseases.
 13. Themethod according to claim 12, wherein said cancer is selected from thegroup consisting of squamous cell cancer of the head-and-neck region,breast cancer and colon cancer.
 14. The method according to claim 12,wherein said hyperproliferative disease is selected from the groupconsisting of psoriasis, myocardial hypertrophy and benign tumor.
 15. Amethod of treating and/or alleviating a disease selected from the groupconsisting of cancer and other hyperproliferative diseases, comprisingthe step of: administering a therapeutically effective amount of apharmaceutical composition comprising an agent, which inhibits KIAA1524.16. The method according to claim 15, wherein said cancer is selectedfrom the group consisting of squamous cell cancer of the head-and-neckregion, breast cancer and colon cancer.
 17. The method according toclaim 15, wherein said hyperproliferative disease is selected from thegroup consisting of psoriasis, myocardial hypertrophy and benign tumor.18. The method according to claim 15 wherein the agent is an antisenseoligonucleotide, a small interfering RNA (siRNA), or a ribozyme.
 19. Themethod according to claim 18, wherein said siRNA is selected from thegroup consisting of SEQ ID NO:s 2 to
 6. 20. The method according toclaim 15, wherein the agent prevents or inhibits cell proliferation andgrowth by inhibiting said KIAA1524 protein from interacting with PP2Acomplex or with the transcription factor c-Myc, or wherein said agentinhibits KIAA1524 proliferative and growth effects that are notdependent on KIAA1524 interaction with PP2A or c-Myc.
 21. The methodaccording to claim 15, wherein the agent is a peptide, a small molecule,an antibody or an aptamer.
 22. The method according to claim 21, whereinsaid peptide comprises at least one amino acid sequence selected fromthe group consisting of SEQ ID NO:s 7 to 30, 32 to 79, and conservativesequence variants thereof.
 23. The method according to claim 22, whereinsaid peptide comprises 1 to 20 consecutive sequences selected from thegroup consisting of SEQ ID NO:s 32 to 79 and conservative sequencevariants thereof.
 24. The method according to claim 22, wherein saidpeptide comprises 1 to 20 consecutive sequences selected from the groupconsisting of SEQ ID NO:s 7 to 30 and conservative sequence variantsthereof.
 25. The method according to claim 15 wherein the agentinactivates the KIAA1524 in the region ranging from aa 461 through 533or any other region on KIAA1524 that participates on interaction betweenPP2A complex and KIAA1524 protein.
 26. A method for determining theinvasiveness of a malignant change in a mammal suspected to suffer fromcancer, said method comprising the steps of: a) assessing the level ofKIAA1524 expression in a sample, suspected to comprise malignant cells,taken from said mammal, b) comparing the expression level from step a)with the expression level of KIAA1524 in a non-malignant control sample,and c) determining said malignant changes as invasive when theexpression level of KIAA1524 in said sample is significantly higher thanthe expression level of KIAA1524 in a non-malignant control sample. 27.The method according to claim 26, further comprising determining saidmalignant changes as gradus III or IV, when the expression level ofKIAA1524 in said sample is more than 2 times higher than in anon-malignant control sample or in non-invasive gradus I and II samples.28. The method according to claim 27, wherein said cancer is coloncancer.
 29. The method according to claim 26, further comprisingdistinguishing invasive tumor types selected from the group consistingof invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC) andIDC with intraductal comedo carcinoma (IDC+ICC), from non-invasive tumortypes when the expression level of KIAA1524 in said sample is more than2 times higher than in a non-malignant control sample.
 30. Apharmaceutical composition comprising a small interfering RNA accordingto claim
 4. 31. A method of inhibiting KIAA1524 in a human or animalpatient in need thereof by administering a therapeutically effectiveamount of a pharmaceutical composition according to claim
 30. 32. Themethod according to claim 31, wherein said patient suffers from adisease selected from the group consisting of cancer and otherhyperproliferative diseases.
 33. The method according to claim 32,wherein said cancer is selected from the group consisting of squamouscell cancer of the head-and-neck region, breast cancer and colon cancer.34. The method according to claim 32, wherein said hyperproliferativedisease is selected from the group consisting of psoriasis, myocardialhypertrophy and benign tumor.
 35. A pharmaceutical compositioncomprising a peptide according to claim
 5. 36. A method of inhibitingKIAA1524 in a human or animal patient in need thereof by administering atherapeutically effective amount of a pharmaceutical compositionaccording to claim
 35. 37. The method according to claim 36, whereinsaid patient suffers from a disease selected from the group consistingof cancer and other hyperproliferative diseases.
 38. The methodaccording to claim 37, wherein said cancer is selected from the groupconsisting of squamous cell cancer of the head-and-neck region, breastcancer and colon cancer.
 39. The method according to claim 37, whereinsaid hyperproliferative disease is selected from the group consisting ofpsoriasis, myocardial hypertrophy and benign tumor.